1. Field of the Invention
This invention resides in the field of electrophoretic separation media, and addresses in particular the elongated gel strips used for isoelectric focusing.
2. Background of the Invention
Electrophoresis for purifying proteins and separating complex protein mixtures has developed to the point where it is now performed in many different ways. Variations arise in the composition of the separation medium, the geometrical configuration of the separation medium, the manner in which mobility through the medium is achieved, and the parameter on which separation is based.
One type of electrophoresis that is particularly useful is isoelectric focusing, in which the proteins are separated in a linear manner according to their isoelectric points. Isoelectric focusing is at times used as the entire separation process, and at other times as the first dimension of a two-dimensional separation, the second dimension being performed by placing the linear medium with its isoelectrically focused zones along one edge of a two-dimensional (xe2x80x9cslabxe2x80x9d-shaped) separation medium. An electric field is then imposed on the two-dimensional medium in a direction transverse to the linear medium, causing migration of the contents of each focused zone out of that medium and into the two-dimensional medium along parallel paths where the contents of each zone are separated according to a parameter other than isoelectric point. The proteins in the original sample thus have the benefit of being separated according to two parameters.
The pH gradient is commonly achieved by using a dimensionally stable medium that consists of a molecular matrix to which functional groups have been attached that are either charged or become charged when the medium is placed in an electric field. Strips of solid media that contain such groups are commonly referred to as xe2x80x9cimmobilized pH gradientxe2x80x9d (xe2x80x9cIPGxe2x80x9d) strips. The composition and structure of these strips are described by Rosengren et al. in U.S. Pat. No. 4,130,470, issued Dec. 19, 1978. The solid material that forms the matrix of the strip is either a granular, fibrous, or membrane material, or a gel. Examples of suitable gels are polyacrylamide, cellulose, agarose, dextran, polyvinylalcohol, starch, and silica gel. The functional groups, which are immobilized on the matrix by covalent bonding or other means, may be positively charged groups such as amino or other nitrogen-bearing groups or negatively-charged groups such as carboxylic acid groups, sulfonic acid groups, boronic acid groups, phosphonic or phosphoric acid groups, or esters of these acids. The typical fabrication procedure for polymeric media is to copolymerize charged or chargeable monomers with uncharged monomers or to include charged crosslinking agents. A monotonic increase or decrease in the concentration of the charged or chargeable groups will produce the desired gradient.
The linear isoelectric focusing medium can be either a tube gel or a flat gel strip, and both have disadvantages. The tube gel must be removed from its tubular enclosure after the first-dimension separation so that the tube gel can be placed in direct contact with the slab gel for the second-dimension separation. Tube gels are difficult to remove from their enclosures, and once removed, are difficult to handle due to their lack of rigidity and the need to minimize contact of the gel with the user""s fingers. Strip gels, by contrast, are relatively easily to manipulate since they are not run inside enclosures and are cast over a plastic backing sheet which the user can handle without touching the gel. Nevertheless, strip gels are generally supplied in dehydrated form and must be rehydrated before use. This is generally accomplished with the sample itself (in diluted form) by simply wetting the strip with the sample and allowing both to stand until equilibration is achieved. Among the limitations of this procedure are that the amount of sample that can be absorbed by the gel in this manner is limited by the diffusion process. In addition, care must then be taken to assure that the strip does not lose water to the atmosphere and thereby become dehydrated. To prevent this from happening, the strip is covered with an electrically insulating, water-immiscible liquid such as mineral oil, and kept covered during the isoelectric focusing. During this time, contact of the two ends of the strip with electrodes must be maintained. In addition, once the isoelectric focusing is completed and before the second-dimension separation is begun, the mineral oil must be removed completely since any residual oil may interfere with the electrical continuity between the strip and the slab gel. Removal of the oil can be a difficult and messy procedure. Finally, the equipment used to perform isoelectric focusing on a strip gel is relatively complicated and expensive owing to the configuration of the gel, the manner in which the gel is placed on the equipment, and the manner in which the electrical contacts are made.
The present invention resides in an assembly in which an isoelectric focusing (IEF) gel can be cast and used, the assembly offering the benefits of both a tube gel and a flat IEF strip and none of the disadvantages. The assembly consists of a rod-shaped casting mold split longitudinally into two halves or half casting molds, each having a half-circle profile so that when the halves are combined along their flat contacting surfaces, the completed mold is tubular in shape with a circular cross section. Grooves or indentations are cut into each of the two contacting surfaces, the grooves in direct opposition to one another to form a chamber of rectangular cross section extending the full length of the mold and open at both ends. One groove receives a flexible backing strip for the gel and the interior of the other, when the two halves are combined, serves as the casting chamber for the gel. The gel once cast in this chamber with the backing strip in place will adhere to the backing strip which can be grasped to remove the gel from the mold.
The circular cross section of the exterior of the combined halves of the assembly permits the assembly to be used in electrophoresis apparatus that is designed for a tube gel, i.e., laboratory instrumentation that is designed to receive a tube gel in its tubular enclosure and to provide the electrical connections to the tube gel that are necessary for electrophoresis to take place. The tube gel shape also permits the gel to be loaded with sample by the enhanced procedures that are used with tube gels, thereby permitting greater sample quantities to be loaded and sample loading to be accomplished in shorter periods of time. The split-mold construction then enables the user to open the assembly after isoelectric focusing is completed and to easily remove and reposition the gel without touching the gel.
In preferred embodiments, the circular cross section extends less than the full length of the assembly, occupying instead only a segment beginning at one end of the assembly, the remaining length being a rectangular cross section to facilitate the sue of screws, clamps, or other means to secure the two halves together in a readily removable manner.
Further features, embodiments, advantages, and implementations of the invention will become apparent from the description that follows.